Coherent anti-Stokes Raman scattering (CARS) is a nonlinear four-wave mixing process that is used to enhance the weak (spontaneous) Raman signal. In the CARS process a pump laser beam (at frequency pump) and a Stokes laser beam (at Stokes) interact, producing an anti-Stokes signal at frequency CARS = 2pump - Stokes. The Stokes beam (Stokes) is typically provided by the 1064nm line from a Nd:Vanadate laser, which also acts as the pump source for an optical parametric oscillator (OPO), while the output from the OPO (680-1010nm) acts as the pump beam (pump). When the frequency difference (beat frequency) between the pump and the Stokes beams matches the frequency of a (Raman active) vibrational mode, such as the CH2 symmetric stretching mode at ~2800 cm-1, the molecular oscillators are coherently driven. This results in an enhanced anti-Stokes (shorter-wavelength) Raman signal that is the basis for the increased vibrational contrast of CARS microscopy.
Figure 1: Coherent anti-Stokes scattering (CARS) energy diagram and schematic of the experimental setup. CARS microscopy
Two fields that have greatly benefited from the development of CARS microscopy are cell biology and tissue imaging. Typically, cell interrogation is performed using fluorescence microscopy. With CARS it is possible to perform chemically specific, label-free imaging at the sub-micron scale. To date CARS microscopy has shown its potential in studies of lipid metabolism, organelle transport, and drug diffusion (pharmaco-kinetics) in living tissue. CARS microscopy has also found use in clinical applications and fast, video-rate imaging of tumor masses in healthy brain tissue has been demonstrated.
Figure 2: CARS microscopy images of cholesteryl palmitate from a fixed tissue sample. The image on the left was obtained using an emission filter commonly used in CARS microscopy experiments. The image on the right was obtained using Semrock part FF01-625/90 and shows enhanced image contrast over the same sample ROI. The same laser intensities and PMT settings were used when acquiring these images.
Image courtesy of Prof. E. Potma (UC Irvine).